PROJECT SUMMARY Excessive TRPM7 channel activity is linked to neuronal cell death, cancer cell metastasis, and as our preliminary data will demonstrate, the development of cardiac fibrosis in a hypertensive hypertrophy/heart failure mouse model. Collectively, these findings underscore a critical role for TRPM7 in the pathology of a multitude of diseases, making channel an attractive target for therapeutic intervention. However, the specific mechanisms controlling TRPM7 activity in vivo remain unknown. We have made the critical discovery that TRPM7 binds to CNNM proteins (CNNM1-4), which our preliminary data indicate function as regulatory subunits of the channel. We further show that PTP4A phosphatases activate TRPM7 in a CNNM-dependent manner. TRPM7 is the first identified ion channel to possess a kinase domain, the function of which is poorly understood. We recently reported the discovery that auto-phosphorylation of the channel plays a decisive role in controlling the stability of TRPM7 protein expression and the channel's localization in cells. We hypothesize that PTP4A phosphatases, CNNMs, and channel phosphorylation operate in concert to regulate TRPM7. In the multi-PI proposal, we propose three specific aims to elucidate the molecular mechanisms controlling the TRPM7 channel with the long-term goal of understanding how the channel becomes upregulated during cardiac fibrosis. In specific aim 1, we will employ electrophysiology, imaging, and biochemical approaches to elucidate the regulation of TRPM7 by CNNMs and PTP4As. In specific aim 2, we will apply analytical mass spectrometry, biochemical, and imaging approaches to understand how phosphorylation of the channel regulates TRPM7 protein expression and its cellular localization. In specific aim 3, we will investigate the specific mechanism(s) controlling pathological stimulation of the channel during cardiac fibrosis. There is an urgent need for new treatments for stroke, cancer, and heart disease, which kill or severely disable millions of individuals each year. Results from our investigation will have a significant impact by uncovering the mechanisms controlling the channel, which may lead to novel clinical approaches for blocking TRPM7's pathological actions in these devastating diseases.